PROJECT SUMMARY Glutamate excitotoxicity causes neuronal apoptosis and necrosis in a myriad of age-associated neurologic conditions such as stroke and Alzheimer’s disease. High dose glutamate stimulation of neurons causes a rapid loss of dendritic spines (DS), membranous protrusions that bud off dendrites. DS are critical for learning and memory, but the structural changes that result in this loss remain poorly understood due to their small size. Up to 10% of DS are dually innervated with inhibitory synapses (DiDS) and found to be more stable than singly innervated DS (SiDS) containing only an excitatory synapse. A process termed compartmentalization is also considered key to DS stability, whereby mature spines with large heads and narrow necks restrict molecules and ions from diffusion into and out of the dendrite. Recent evidence suggests an actin diffusion barrier within the DS neck and head-neck junction could be key to compartmentalization, but this remains poorly understood. Following excitation calcium influx causes actin network remodeling that drives DS morphologic change. Inhibitory synapses on DiDS have been found to dampen excitatory post synaptic potentials and calcium influx, and upwards of 86% contain a spine apparatus. Based on these findings, I hypothesize following glutamate excitotoxicity DiDS maintain a more stable DS structure than SiDS. To investigate I will use high resolution cryo-electron tomography paired with correlative fluorescence to compare DiDS and SiDS and elucidate structural changes that result in DS loss following glutamate excitotoxicity. Aim 1 will determine the ultrastructural differences between DiDS and SiDS actin networks under normal conditions. Aim 2 will determine the ultrastructural changes that occur between DiDS and SiDS following glutamate excitotoxicity. If successful, this project would determine what high resolution structural differences exist between DiDS and SiDS and whether DiDS are more stable following excitotoxic shock. This would also provide investigators of excitotoxicity high resolution structural evidence for why inhibitory synapses could be therapeutic targets to prevent neuronal injury.